The present invention relates to the production of covalently closed circular (ccc) recombinant DNA molecules. Such molecules are useful in biotechnology, transgenic organisms, gene therapy, therapeutic vaccination, agriculture and DNA vaccines.
With the invention in mind, a search of the prior art was conducted. E. coli plasmids have long been the single most important source of recombinant DNA molecules used by researchers and by industry. Today, plasmid DNA is becoming increasingly important as the next generation of biotechnology products (gene medicines and DNA vaccines) make their way into clinical trials, and eventually into the pharmaceutical marketplace. Plasmid DNA vaccines may find application as preventive vaccines for viral, bacterial, or parasitic diseases; immunizing agents for the preparation of hyper immune globulin products; therapeutic vaccines for infectious diseases; or as cancer vaccines. Plasmids are also utilized in gene therapy or gene replacement applications, wherein the desired gene product is expressed from the plasmid after administration to the patient.
The basic methods for obtaining plasmids (by bacterial fermentation), and for their purification (e.g., by the alkaline lysis method (Birnboim, H C, Doly J. 1979, Nucleic Acids Res. 7: 1513-1523)) are well-known.
The use of reduced growth rate is the unifying principle in high quality, high yield plasmid fermentations. The optimal temperature for E. coli growth is 37° C. However, lower temperatures (30-37° C.) may be used in batch fermentation to cause a reduced maximum specific growth rate. Higher temperatures (36-45° C.) can also be employed to induce selective plasmid amplification with some replication origins such as pUC (Lin-Chao S, Chen W T, Wong T T. 1992 Mol. Microbiol. 6: 3385-3393), and pMM1 (Wong E M, Muesing M A, Polisky, B. 1982 Proc Natl Acad Sci USA. 79: 3570-3574) (Reviewed by Carnes A E. 2005 BioProcess International 3: 36-44).
The fermented bacterial cell paste is then resuspended and lysed (using a combination of sodium hydroxide and sodium dodecylsulfate), after which the solution is neutralized by the addition of acidic salt (e.g., potassium acetate), which precipitates the bacterial DNA and the majority of cell debris. The bulk of super-coiled plasmid DNA remains in solution, along with contaminating bacterial RNA, DNA and proteins, as well as E. coli endotoxin (lipopolysaccharide, or LPS). The soluble fraction is then separated by filtration and subjected to a variety of purification steps, which may include: RNase digestion; chromatography (ion exchange gel filtration, hydroxyapatite, gel filtration, hydrophobic interaction, reverse phase, HPLC, etc.); diafiltration; organic extraction, selective precipitation, etc.
Today, the FDA standards are not defined except in preliminary form (see: FDA Points to Consider on Plasmid DNA Vaccines for Preventive Infectious Disease Indications, 1996). However, in the future, international standards for plasmid DNA purity are likely to be the same or very similar to those that are used for recombinant protein products similarly produced from E. coli fermentation, and such standards exceed the current purity attainable from established methods. Most glaringly, the accepted standard of <100 pg host genomic DNA per dose (see: FDA Points to consider in the characterization of cell lines used to produce biologics, 1993) is far below the levels currently attainable for purified plasmid preparations (100 pg per 1 mg dose is equivalent to one part per ten million).
Clearly, increasing the purity of the starting material through improved yield would improve the final product purity, and ultimately is an important goal to facilitate the manufacture of clinical grade DNA on an industrial scale.
High Copy Replication Origins
Circular plasmids of bacteria replicate by theta, strand displacement or Rolling circle mechanisms (see Del Solar G, Giraldo R, Ruiz-Echevarria, Espinosa M, and Diaz-Orejas R. 1998 Microbiol. Molec. Biol. Reviews 62: 434-464 for a comprehensive review).
To date, all therapeutic plasmids (e.g. for gene therapy or vaccination) utilize theta replication origins. Theta replication requires synthesis of a primer RNA, and DNA synthesis is initiated by extension of the RNA primer. This type of replication is characterized by the separation of the DNA strands at the origin creating a characteristic theta-shaped replication bubble.
Most theta replication plasmids such as R6K (used in pCOR therapeutic plasmid), R1 or pKL1 (but not pMB1 or ColE1 derived; see below) require a plasmid-encoded Rep initiator protein which binds to specific sequences (often tandem direct repeats called iterons) in the replication origin and recruit host DnaA initiator protein to flanking dnaA box. In contrast, pMB1 and ColE1 plasmids produce a RNAII primer that is cleaved by RNase H, and extended by DNA polymerase I prior to switching to DNA polymerase III.
Origin Biology: ColE1 Type Origin
The vast majority of therapeutic plasmids currently in use are derived from pBR322 or pUC plasmids, and use high copy derivatives of the pMB1 origin (closely related to the ColE1 origin). For example, pcDNA3, VR1012, pVAX1, pVC0396, DNA vaccine plasmids utilize the pMB1 derived pUC origin, pVC0396 uses the ColE1 derived pMM1 origin, while the pCMVkm2 DNA vaccine plasmid utilizes the pMB1 derived ROP-pBR322-derived origin. The ColE1 type (ColE1) plasmid copy number is controlled by RNAI, an antisense RNA (FIG. 1; reviewed in Del Solar et al, Supra, 1998). RNAI forms a tRNA like structure, with three stem loops. The 5′ end of the primer, RNAII, is complementary to RNAI and forms a three loop structure antisense to RNAI. The RNAI interaction initiates at the complementary unpaired loops in RNAII (kissing interaction; reviewed in Wagner E G H, Brand S. 1998 TIBS 23: 451-454) preventing its maturation into the replication primer. The loop regions contain YUNR (Y=pyrimidine R=purine) motifs that specify two intraloop hydrogen bonds forming U-turn structures. This motif is present in the loops in natural antisense RNA regulated systems including RNAI and RNAII, and is speculated to be an enhancer of RNA pairing rates (Franch T, Petersen M, Wagner E G H, Jacobsen J P, Gerdes K. 1999 J Mol Biol 294:1115-1125). Analysis of copy number mutants isolated in the RNAI/RNAII overlap region indicates the importance of at least loops 2 and 3 in repression (Moser D R, Campbell J L. 1983 J Bacteriol 154: 809-818). The RNAI/II interaction is stabilized, and potentially protected from RNase degradation by the repressor of primer (ROP or ROM) protein and destabilized by uncharged tRNAs (see below). There is a short window of opportunity for the RNAI/II interaction, as RNAII changes conformation as it forms the primer, becoming inaccessible to RNAI. Maturation of the primer requires RNaseH cleavage, and primer extension by DNA polymerase I before switching to DNA polymerase III (FIG. 1). RNAI is constitutively synthesized to high levels relative to RNAII, but is degraded rapidly. RNAI inhibition is efficient; it is estimated that in the wild type origin (i.e. pBR322) at most 5% of RNAII transcripts are processed into an active primer (Lin-Chao S, Bremer H. 1987 J Bacteriol. 169:1217-1222).
The rate of processing and degradation of RNAI regulates ColE1 plasmid copy number (Lin-Chao S, Cohen S N. 1991. Cell 65:1233-1242). RNase E is the primary endogenous RNase that degrades RNAI to the unstable RNAL5 (FIG. 1). Polyadenylation of RNAI by polyA polymerase (pcnB gene product) reduces affinity for RNAII (Xu F F, Gaggero C, Cohen S N. 2002 Plasmid 48:49-58) and enhances RNAI degradation by RNase E (Xu F, Lin-Chao S, Cohen S N. 1993 Proc. Natl. Acad. Sci. 90:6756-6760). RNaseIII also contributes to RNAI degradation (see Binnie U, Wong K, McAteer S, Masters M. 1999 Microbiology 145:3089-3100 for a review of RNAI degradation pathways) as does polynucleotide phosphorylase.
High Copy Number Replicons:
Inducible plasmid copy number replication origins are preferred for therapeutic plasmids. This is because constitutive high plasmid levels increases metabolic burden on the cells, and may result in instability, lower productivity or toxicity to cells. Mechanisms for creation of conditional high copy plasmids, and conditional runaway replicons, are discussed below.
ColEI type origin: Numerous copy up versions of the ColEI and pMB1 origins have been isolated, as either spontaneous or selected mutations (in screens for temperature sensitive resistance to extreme levels of antibiotic). The lesions associated with several high-copy number mutants are clustered in the RNAI promoter but do not affect RNAI transcription. Rather, they appear to affect the secondary structure of the RNAII replication primer that may either affect RNAI/RNAII interactions or the ability of RNAI to inhibit replication initiation (Lin-Chao et al, Supra, 1992; Fitzwater T, Zhang X, Bible R, Polisky B. 1988 EMBO J. 7:3289-3297; Gultyaev A P, Batenburg F H D, Pleij C W A. 1995 Nuc Acids Res 23:3718-3725). Commonly utilized copy up derivatives of the pMB1 origin (e.g. pUC19; Lin-Chao et al, Supra, 1992) or ColE1 origin (pMM1, pMM7; Wong et al, Supra, 1982) delete the accessory ROP (rom) protein and have an additional alteration that destabilizes the RNAI/RNAII interaction. For temperature sensitive origins (e.g. pUC, pMM1, pMM7), shifting of the culture from 30 to 42° C. leads to 30-40 fold increase in plasmid copy number to 300 copies per cell. Many of these derivatives are maximally induced by both temperature and entry into stationary phase (pMM1, pMM7, pUC; Lin-Chao et al, Supra, 1992; Fitzwater et al, Supra, 1988; Wong et al, Supra, 1982). pMM7 is reported to have 119 copies per cell in early log, and undergo a further 21 fold increase in copy number by late stationary phase. In stationary phase, pMM7 plasmid DNA accounts for >50% of total cell DNA (Fitzwater et al, Supra, 1988).
Other pMB1 copy number mutations have been identified. For example, the pXPG mutation changes the −10 region of the RNAII promoter from TAATCT to TAATAT, which may increase the expression of RNAII (Bert A G, Burrows J, Osborne C S, Cockerill P N. 2000 Plasmid 44:173-182). This increases the copy number of pBR322 at low temperature. Derivatives that over-express RNAII conditionally at high temperature (using an inducible promoter) have been rationally designed to increase copy number. For example, dual origin plasmids with ColE1 RNAII expression driven by λPR promoter increase plasmid copy number from 3-4 at 30° C. to 200-300 at 42° C. (Wright E M, Humphreys G O, Yarranton G T. 1986 Gene 49: 311-321; Yarranton, G T, Humphreys, G O, Robinson, M K, Caulcott, C A, Wright E M. 1991 U.S. Pat. No. 5,015,573).
A nonconditional mutation at the 3′ end of RNAI in pBR322 results in a copy number of 1000 per cell or 65% of total cellular DNA; this mutation is also associated with toxicity, due to its constitutive nature (Boros I, Posfai G, Venetianer P. 1984 Gene 30:257-260; Boros I, Venetianer P, Posfai G. 1984 U.S. Pat. No. 4,703,012).
As well, a pBR322 plasmid was modified to produce a truncated RNAII driven by a inducible synthetic promoter (in addition to native RNAII); this increased the copy number of pBR322 four fold upon induction, possibly due to inhibition of RNAI by the truncated RNAII (Bachvarov D, Jay E, Ivanov I. 1990 Folia Microbiol 35:177-182).
Alterations to RNAI and RNAII loop 2 by random mutagenesis, followed by selection for colonies exhibiting high levels of antibiotic resistance, was used to isolate high copy number variants of pET11a-SOD (pBR322 origin). These variants exhibit 14 fold increase in copy number, which is further increased up to 2500 plasmids/cell by IPTG induction of the T7 promoter driven SOD gene (Bayer K, Grabherr R, Nisson E, Striedner G. 2004 U.S. Pat. No. 6,806,066B2). The uninduced increases may be due to decreased affinity in RNAI/RNAII binding due to elimination of the RNA/RNA interaction enhancing YUNR U-turn structures. The induction after IPTG addition may be due to protein synthesis inhibition, and therefore be similar to copy number amplification after chloramphenicol treatment (Teich A, Lin H Y, Andersson L, Meyer S, Neubauer P. 1998 J Biotechnol 64:197-210).
A major disadvantage with all these modified high copy replicons is that they cannot be utilized to improve yield of existing plasmids without reengineering. Retrofitted plasmids would require reevaluation of the biological activity and safety, since new sequences may alter the immunogenicity or expression of a target gene from the plasmid.
Increasing Plasmid Copy Number without Alteration to Target Plasmid Sequences
Host strains with inducible factors that cause increased replication could improve productivity by a synergistic interaction with existing high copy ColE1 origins. Theoretically, such factors could participate in the plasmid replication pathway (FIG. 1) at several steps including:                1) Increase RNAI degradation (e.g. increased poly A polymerase or specific mRNA degradosome components such as RNaseE),        2) Inhibit RNAI interaction with RNAII (e.g. uncharged tRNA's)        3) Enhance primer formation (e.g. stabilize R loops; see below)        4) Enhance primer processing (e.g. increased RNaseH),        5) Increase initial DNA synthesis rates (e.g. increased DNA polymerase I)        
Overexpression or repression of E. coli gene products on their own might not increase copy number due to the complexity of the regulatory pathways for each of these genes:                gene product may be saturating, or not a limiting component of a complex        gene product may by redundant        gene product may be toxic at low or high levels or        gene product overexpression may induce a compensatory effect.        
For example, as expected, ColE1 plasmid copy number is decreased in RNaseE or PolyA polymerase mutants. However, increased polyadenylation by overexpression of pcnB, although increasing levels of RNaseE, does not increase plasmid copy number (Mohanty B K, Kushner S R. 2002 Mol Micro 45:1315-1324). Inactivation of genes involved in factor-dependent transcription termination, nusG or rho, leads to runaway lethal replication of ColE1 plasmids including pUC; the excess chromosomal R-loops in these strains are hypothesized to titrate R-loop destabilizing factors (i.e. possibly rnhA, RecG, DNA topoisomerase I, RNaseH; Harinarayanan R, Gowrishankar J. 2003 J Mol Biol 332:31-46). However, the quality of plasmids induced in this way may be altered. Topoisomerase is required to prevent plasmid knotting (Shishido K, Komiyama N, Ikawa S. 1987 J Mol Biol 195:215-218) remove R-loops (Masse E, Drolet M. 1999 J Biol Chem 274:16659-16664) and regulates plasmid supercoiling. As expected, plasmid DNA supercoiling is reduced in rho mutants (Fassier J S, Arnold G F, Tessman I. 1986 Mol. Gen. Genet. 204:424-429). In general, plasmid quality may be compromised with alteration of gene products involved in DNA replication, repair, recombination, supercoiling, or other pathways that are essential for plasmid integrity. Such systems may also self limit yield due to secondary toxicity (e.g. pcnB or RNaseE overexpression is toxic to the cell).
A variety of screens for copy number enhancing mutations have been performed, and yielded chromosomal mutations that increase plasmid copy number. Some of these mutations are speculated to alter antisense regulation of the plasmid in favor of higher copy number (for example, see Tao L, Jackson R E, Rouviere P E, Cheng Q. 2005 FEMS Microbiol Lett. 243: 227-233). However, these screens have all been performed with relatively low copy number pBR322 based plasmids, and the utility of the identified mutations to increase copy number of higher copy number plasmids such as pUC have not been demonstrated.
Antisense regulation of copy number by uncharged tRNAs has been observed. Amino acid starvation leads to expression of the stringent (wild type) or relaxed (relA) response. In relA strains, uncharged tRNAs are elevated under conditions of amino acid limitation such as in starved stationary phase culture. Binding of the uncharged tRNAs to loops in RNAI and/or RNAII prevents RNAI interaction with RNAII, and increases plasmid copy number. This can increase copy number up to 10 fold in amino acid starved stationary phase cells versus log phase cells (pBR322 is amplified from 50 to 340; pUC9 is amplified from 90 to 940; Schroeter A, Riethdorf S, Hecker M. 1988 J Basic Microbiol 28:553-555). However, copy number is also increased in stationary phase cells by supplementation (rather than depletion) of amino acids (Angelov I, Ivanov I. 1989 Plasmid 22: 160-162), and is observed in RelA+ strains, so copy number increase in stationary phase is complex, with additional unknown mechanisms to increase copy number.
Stationary phase amplification due to amino acid depletion has been exploited to increase pBR322 yield in fermentation culture by depletion of arginine during batch or fed-batch growth (Hofinann K H, Neubauer P, Riethdorf S, Hecker M. 1990 J Basic Microbiol 30:37-41). However, the yield increase was modest (only 50 mg/L), was shown only with a moderate copy number plasmid (pBR322) and is not a generally useful process, since current optimal plasmid DNA fermentation processes that yield >100 mg plasmid/L use growth restriction to produce high levels of plasmid prior to stationary phase (Reviewed in Carnes, Supra, 2005).
Uncharged tRNA induction of plasmid copy number has been hypothesized to be through binding interaction with either the anticodon, dihydrouridylic loop (these interactions must be stabilized by an unknown mechanism in uncharged tRNAs) or 3′ CCA-OH sequence of the tRNA, with complementary sequences in RNAI and RNAII loops (Yavachev L, Ivanov I. 1988 J. Theor. Biol. 131:235-241; Wang Z, Le G, Shi Y, Wegrzyn G, Wrobel B. 2002 Plasmid 47:69-78; reviewed in Wegrzyn G. 1999 Plasmid 41:1-16). Removal of the homology to tRNAs in RNAI and RNAII loop 2 by site directed mutagenesis eliminated copy number variation; this demonstrates that loop 2 is a key target (Grabherr R, Nilsson E, Striedner G, Bayer K. 2002 Biotechnol Bioeng. 2002; 77:142-147).
Overexpression of a naturally occurring antisense sequence may be responsible for repressing the copy number defect of a pBR322 plasmid in a pcnB mutant by interfering with RNAI activity through loop III (Sarkar N, Cao G, Jain C. 2002 Mol Genet Genomics 268: 62-69). However, the utility of this putative antisense regulator was not determined since induction of plasmid copy number was not demonstrated in a wild type background, nor was the method shown to have application for the production of high copy plasmids. A combinatorial selection scheme (using a low copy number reporter plasmid that produced a low level of a selective agent that was insufficient for cell survival, but increased levels would allow cells to grow) using random RNA expression plasmids identified an RNA that increased INcFII plasmid copy number (Ferber M J, Maher III J. 1998 J Mol Biol. 279:565:576); however, this methodology was not applied to improve copy number of high copy plasmids, or of ColE1 plasmids.
Collectively, none of the host strains or methods described above have been shown to have utility in improving yield of existing high copy plasmids.
Current Barriers
Many therapeutic plasmids are not inducible to maximal levels or contain detrimental sequences that limit plasmid copy number, limiting productivity yields. This may be overcome by increasing plasmid replication rate. Selection of new inducible replication origins that reach maximal levels in log phase (rather than late stationary phase) would be applicable for fermentation processing. However, a major disadvantage with engineering new runaway replicons is that they cannot be utilized to improve yield of existing plasmids without reengineering. Retrofitted plasmids would require reevaluation of the biological activity and safety, since new sequences may alter the immunogenicity or expression of a target gene from the plasmid.
Even in view of the prior art, there remains a need for a cost effective method to further increase plasmid DNA production. Fermentation processes which incorporate what is currently known in the art to improve plasmid productivity, such as reduced growth rate and plasmid copy number induction with high temperature can give yields exceeding 1 gm plasmid DNA/L with optimal high copy pUC origin plasmids (Reviewed in Carnes, Supra, 2005). Further yield increases would further reduce costs and improve final product purity. Moreover, alternative origins (e.g. pBR322 origin with ROP deletion) or non optimal pUC origin-containing plasmids may produce 1 or 2 orders of magnitude lower levels of plasmid. This low yield imposes a cost and purity burden on commercialization of plasmid DNA production processes using these vectors. Although economies of scale will reduce the cost of DNA significantly in the future, a far more economical solution to this problem is needed in order to achieve the desired cost. As well, international standards for plasmid DNA purity are likely to be the same or very similar to those that are used for recombinant protein products similarly produced from E. coli fermentation, and such standards exceed the current purity attainable from established methods. Increasing the yield (mg of DNA/gram of cell paste) in fermentation would both decrease the cost and increase the purity of the DNA (because it reduces the amount of material being processed).